Scientists and industry alike are continually seeking methods to evaluate molecular interactions and eliminate the uncertainty associated with utilizing labels to detect the location of molecules of interest. Label-free technologies represent a holy grail in terms of addressing this issue, as these techniques allow researchers to look at molecular systems without perturbing them with extraneous chemistries that fundamentally change the dynamics of interaction. The sensitivity of instruments designed to analyze these molecular interactions is of paramount concern because often molecules of interest are difficult and expensive to produce and/or isolate, or are present in biological samples only at very low concentrations. Compounding the issue of miniscule quantities are the numerous variations of analytes, such as in drug development's combinatorial chemistry libraries of which binding characteristics are desired, which means that viable technologies applied to this field must provide array capabilities that can be integrated with high throughput screening methods. It must also be sensitive enough to detect precious amounts of interesting molecules, quickly and specifically.
The optical sensors based on the detection of analyte binding to thin receptor films at the sensor surface have been studied intensively. The use of an optical Total Internal Reflection (TIR) configuration for measurement of index of refraction changes in the evanescent field is common to interferometers, elipsometers and polariscopes. All these techniques can be applied to the measurements of chemical or biological layered media, with the goal of label free detection.
Internal reflection ellipsometry (IRE) has been used in earlier studies on orientation of liquid crystals and absorption of solutes onto substrate surfaces and for measurements of the refractive index of liquids. In these studies, however, measurements were conducted in the total internal reflection region of the incident angle that is not favorable for thickness measurements owing to low sensitivity. Therefore, in an effort to enhance the sensitivity of such thickness measurements, many groups successfully pursued the implementation of the Surface Plasmon Resonance (SPR) effect, a method falling under the more general Frustrated Total Internal Reflection (FTR) approach. However, there remains a need for highly sensitive devices for measurements of both thickness and index of refraction changes in bio- and chemical sensing devices.
Several methods have been employed to measure the spatial reflection coefficients and overall intensity from the sensor interface. These intensity-based techniques suffer from the fluctuation of intensity in light sources and the relatively small-reflected coefficient from the sensor surface. Higher detection sensitivity is always desirable for improving sensing performance. It has been found that a lightwave's phase can change much more abruptly than the intensity when the refractive index or thickness of a binding layer on the surface has been changed. Several methods have been employed in sensors by measuring the phase change from the sensor interface during SPR, even with the capability of sensor array imaging. Recently, a sensor based on the combination of SPR and heterodyne interferometry with extremely high sensitivity and low-noise was proposed.
It is commonly known that during both TIR and FTR conditions the phase difference between p- and s-polarized components of reflected beam experiences a rapid shift whenever the optical properties of an adjacent medium change, such as refractive index or thickness of the affinity sensitive layer. It was also shown that measurement of the phase shift of the p-polarized component of the incident beam yields significantly higher sensitivity than SPR techniques that measure intensity associated with incident angle change. Furthermore, the phase-change method allows using both metal coated and optically transparent transducers without a special metallic coating.
Although the proposed method is the same for both TIR and FTR conditions in terms of measured parameters and general configuration of the system, there are some additional considerations and effects that take place when using the FTR approach. Utilizing the SPR phenomena in sensing applications has been demonstrated in several different configurations. A common approach uses the Kretschman configuration. A coherent p-polarized optical wave is reflected under TIR conditions on an interface between an optically dense material, such as a glass prism, and a rarefied medium, which in this case is the sample medium, whose index of refraction is lower than the dense medium. The interface between the two media is coated with a thin conductive metal film, which acts as an absorber for the optical wave. When specific conditions dependent on the light wave's angle of incidence, wavelength and the media's refractive indices are met, the optical wave causes the metal's surface plasmon electrons to oscillate at resonance, absorbing the wave's energy in the metal film. During these resonance conditions, variations in the sample's index of refraction will produce sharp changes to the optical phase of the p-polarized component, while the s-polarization phase remains relatively constant. At the resonant conditions, most of the p-polarization light component in contrast to TIR condition is absorbed in the metal film via the SPR effect. This fact is exploited by intensity based SPR sensors and elipsometers, relating the conditions of the intensity minimum of the reflected light to the optical configuration to thereby deduce the sample's index of refraction or layer thickness.